内皮细胞抑制素血管内皮细胞生长抑制因子_(151)融合基因治疗胃癌的作用及其机制

Inhibitory mechanisms of endostatin-vascular endothelial growth inhibitor_(151) chimeric gene therapy on gastric carcinoma neovascularization

目的探讨新型内皮细胞抑制素血管内皮细胞生长抑制因子151融合基因(hENDOVEGI151)治疗胃癌的作用机制。方法应用重复感染系数(MOI=100)的重组腺病毒AdhENDOVEGI151转染胃癌SGC7901、MKN28细胞和血管内皮ECV304细胞4h后,继续培养6d,噻唑蓝(MTT)比色法检测第1至6天3种细胞的存活率;流式细胞仪(FCM)丙化碘锭(PI)单染色法检测转染后48h胃癌和内皮细胞凋亡的情况;应用DNA片断化试验分析转染后12、24、36、48h时ECV304凋亡情况;应用逆转录聚合酶链反应(RTPCR)和蛋白印迹法(Westernblot)检测融合基因转染对SGC7901表达促血管生成因子VEGF165的影响。结果AdhENDOVEGI151治疗强烈抑制ECV304增殖,72h抑制率为55.18%,144h抑制率89.86%;FCM检测出现明显凋亡峰,凋亡细胞约占(20.70±5.83)%,并且出现G1期阻滞(65.41±2.38)%和S期明显减少(21.81±1.52)%,与AdLacZ组和对照组比较差异有统计学意义(P<0.01);转染组细胞DNA出现典型的梯形条带,尤以转染后24～36h最为明显,AdLacZ组及对照组DNA无裂解。AdhENDOVEGI151转染对胃癌细胞无直接毒性作用,但明显下调胃癌细胞VEGF165的表达水平。结论AdhENDOVEGI151治疗一方面强烈抑制内皮细胞增殖,诱导凋亡;另一方面抑制胃癌细胞表达VEGF165,多角度联合抑制肿瘤新生血管形成,使肿瘤细胞因缺血而发生大量凋亡。

Objective To study the inhibitory mechanisms of endostatin-vascular endothelial growth inhibitor151 （hENDO-VEGI151） chimeric gene therapy on gastric carcinoma neovascularization. Methods Recombinant adenovimses Ad hENDO-VEGI151 was used to treat human vascular endothelial cell line ECV304, human gastric carcinoma cell line SGC-7901 and MKN-28. Fusion protein expression of Ad hENDO-VEGI151 in vitro was identified by Western blot. MTT assay was used to determine the SGC-7901, MKN-28 and ECV-304 cells growth rate. Apoptosis was analyzed by FCM and detection of DNA fragmentation. RT-PCR and Western blot were employed to investigate VEGF165 expression in gastric cancer cells. Results Western blot showed that the molecular weight of fusion protein was about 41kD after infection of ECV-304, SGC-7901 and MKN-28 cells with supernatant of Ad hENDO-VEGII51. No changes in the growth rate and DNA content of SGC-7901 were found. However, Ad hENDO-VEGI151 showed a specific inhibition on the proliferation of ECV-304 cells, and the inhibition rate reached 55.18 % in 72 h and 89.86% in 144 h, respectively. A sub-G1 peak was detected in Ad hENDO-VEGII51 treated ECV-304, and percentage of apoptosis cells was 1.51% of Ad LacZ treated, 1.83% of controls and 20.7 % of Ad hENDO-VEGI151 treated respectively, with cells growth arrest in G1 phrase of the cell cycle. DNA of Ad hENDO-VEGI151 treated ECV-304 showed a typical DNA ladder at 24-36 h after infecting. An obvious down-regulation of VEGF16s expression in cancer cells was identified by RT-PCR and Western blot. Conclusion The fusion gene therapy can directly affect endothelial cell functions by inhibiting the proliferation and inducing apoptosis; On the other hand, it can decrease VEGF165 production by tumor cells or endothelial cells and stromal cells. Our findings strongly suggest that the fusion combines two potent anti-angiogenic genes to increase the suppression of tumor angiogenesis. Fusion protein, operating through different mechanisms, may result in synergistic effects.